食品科学

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限制性内切酶酶切确证河豚鱼成分PCR检测结果

曲良苗1,2,陈文炳1,3,*,缪婷玉1,3,邵碧英1,3,彭 娟1,3,江树勋1,3   

  1. 1.福建出入境检验检疫局检验检疫技术中心,福建 福州 350001;2.福建农林大学食品科学学院,
    福建 福州 350003);3.福建省检验检疫技术研究重点实验室,福建 福州 350001
  • 出版日期:2014-04-25 发布日期:2014-05-13
  • 通讯作者: 陈文炳
  • 基金资助:

    国家质量监督境检验检疫总局科技计划项目(2008IK175; 2013IK149)

Confirmation of PCR Results for Puffer Fish Components by Restriction Endonuclease Digestion

QU Liang-miao1,2, CHEN Wen-bing1,3,*, MIAO Ting-yu1,3, SHAO Bi-ying1,3, PENG Juan1,3, JIANG Shu-xun1,3   

  1. 1. Technical Center of Inpsection and Quarantine, Fujian Entry-Exit Inspection and Quarantine Bureau, Fuzhou 350001, China;
    2. College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350003, China;
    3. Fujian Provincial Key Laboratory of Inspection and Quarantine Technology Research, Fuzhou 350001, China
  • Online:2014-04-25 Published:2014-05-13
  • Contact: CHEN Wen-bing

摘要:

动植物成分的聚合酶链式反应(polymerase chain reaction,PCR)检测往往出现假阳性结果,为了有效排 除河豚鱼成分检测中出现的假阳性现象,提高检测结果的准确性,应用河豚鱼PCR检测引物进行河豚鱼成分的PCR 检测与限制性内切酶NmeA Ⅲ酶切确证实验。18 个供试样品中3 个样品无PCR扩增产物,判为阴性结果,15 个样品 初步判为疑似阳性。应用限制性内切酶NmeA Ⅲ对疑似阳性样品的PCR产物进行酶切与电泳分析,电泳图谱与河豚 鱼阳性对照不同的2 个样品,判定为假阳性结果,电泳图谱与阳性对照相同的4 个未知学名的河豚鱼加工样品,确 证为阳性结果,即检出河豚鱼成分,PCR产物序列经GenBank同源性序列查询比对(BLAST)予以验证,建立了简 便的河豚鱼成分PCR检测结果确证方法。

关键词: 河豚鱼, PCR检测, 限制性内切酶, DNA测序, 结果确证

Abstract:

False positive results frequently happen in PCR detection of animal and plant components. In order to exclude
the possibility of false positive results, restriction endonuclease digestion was used to confirm the results of PCR for
puffer fish components. Eighteen samples were detected, of which 3 samples were sentenced to be negative whereas the
remaining 15 samples were suspected positive based on PCR results. Restriction endonuclease (NmeA Ⅲ) digestion and
agarose gel electrophoresis were used to analyze the PCR products of the suspected positive puffer fish component. The
electrophoresis patterns of PCR fragments digested by NmeA Ⅲ were different from the positive results of puffer fish in 2
non-puffer fish samples, which were judged as false positive. The electrophoresis patterns of 4 samples of processed puffer
fish with unknown scientific names agreed with those of the puffer fish positive samples and were therefore confirmed.
The PCR products were verified by GenBank DNA sequence homology sequence query (BLAST). In conclusion, a
simple method to confirm PCR results of puffer fish ingredients has been established in this study.

Key words: puffer fish, polymerase chain reaction (PCR) detection, restriction endonuclease, DNA sequence, results confirmation

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